Skip to main content
SpringerLink
Log in

Rat Pheochromocytoma PC12 Cells in Culture

  • Protocol
  • First Online:
Chromaffin Cells

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2565))

Abstract

PC12 cells serve as a secretory cell model, especially suitable for studying the molecular mechanisms underlying fusion pore kinetics in regulated exocytosis of dense-core vesicles (DCVs). In this chapter, we describe a series of PC12 cell culture procedures optimized for real-time functional assays such as single-vesicle amperometry. In addition, these conditions have been widely used for single-cell biochemical assays such as the proximity ligation assay with immunostaining.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Levitan IB, Kaczmarek LK (2015) The neuron. Oxford University Press, New York

    Book  Google Scholar 

  2. Wassenberg JJ, Martin TF (2002) Role of CAPS in dense-core vesicle exocytosis. Ann N Y Acad Sci 971:201–209

    Article  CAS  Google Scholar 

  3. Greene LA, Tischler AS (1976) Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci U S A 73:2424–2428

    Article  CAS  Google Scholar 

  4. Martin TFJ (1994) Identification of proteins required for Ca2+−activated secretion. Ann N Y Acad Sci 710:328–332

    Article  CAS  Google Scholar 

  5. Kasai H, Takagi H, Ninomiya Y et al (1996) Two components of exocytosis and endocytosis in phaeochromocytoma cells studied using caged Ca2+ compounds. J Physiol 494:53–65

    Article  CAS  Google Scholar 

  6. Westerink RH, Ewing AG (2008) The PC12 cell as model for neurosecretion. Acta Physiol (Oxf) 192:273–285

    Article  CAS  Google Scholar 

  7. Hay JC, Martin TF (1992) Resolution of regulated secretion into sequential MgATP-dependent and calcium-dependent stages mediated by distinct cytosolic proteins. J Cell Biol 119:139–151

    Article  CAS  Google Scholar 

  8. Wang CT, Lu JC, Bai J et al (2003) Different domains of synaptotagmin control the choice between kiss-and-run and full fusion. Nature 424:943–947

    Article  CAS  Google Scholar 

  9. Wang CT, Bai J, Chang PY et al (2006) Synaptotagmin-Ca2+ triggers two sequential steps in regulated exocytosis in rat PC12 cells: fusion pore opening and fusion pore dilation. J Physiol 570:295–307

    Article  CAS  Google Scholar 

  10. Chow RH, Von Ruden L (1995) Electrochemical detection of secretion from single cells. In: Sakman B, Neher E (eds) Single-channel recording. Plenum Press, New York, pp 245–275

    Chapter  Google Scholar 

  11. Albillos A, Dernick G, Horstmann H et al (1997) The exocytotic event in chromaffin cells revealed by patch amperometry. Nature 389:509–512

    Article  CAS  Google Scholar 

  12. Jackson MB (2007) In search of the fusion pore of exocytosis. Biophys Chem 126:201–208

    Article  CAS  Google Scholar 

  13. Wang CT, Grishanin R, Earles CA et al (2001) Synaptotagmin modulation of fusion pore kinetics in regulated exocytosis of dense-core vesicles. Science (New York, N.Y.) 294:1111–1115

    Article  CAS  Google Scholar 

  14. Zhang Z, Jackson MB (2008) Temperature dependence of fusion kinetics and fusion pores in Ca2+−triggered exocytosis from PC12 cells. J Gen Physiol 131:117–124

    Article  Google Scholar 

  15. Zhang Z, Hui E, Chapman ER et al (2009) Phosphatidylserine regulation of Ca2+−triggered exocytosis and fusion pores in PC12 cells. Mol Biol Cell 20:5086–5095

    Article  CAS  Google Scholar 

  16. Zhang Z, Hui E, Chapman ER et al (2010) Regulation of exocytosis and fusion pores by synaptotagmin-effector interactions. Mol Biol Cell 21:2821–2831

    Article  CAS  Google Scholar 

  17. Zhang Z, Jackson MB (2010) Membrane bending energy and fusion pore kinetics in ca(2+)-triggered exocytosis. Biophys J 98:2524–2534

    Article  CAS  Google Scholar 

  18. Zhang Z, Zhang Z, Jackson MB (2010) Synaptotagmin IV modulation of vesicle size and fusion pores in PC12 cells. Biophys J 98:968–978

    Article  CAS  Google Scholar 

  19. Zhang Z, Wu Y, Wang Z et al (2011) Release mode of large and small dense-core vesicles specified by different synaptotagmin isoforms in PC12 cells. Mol Biol Cell 22:2324–2336

    Article  CAS  Google Scholar 

  20. Chiang N, Hsiao YT, Yang HJ et al (2014) Phosphomimetic mutation of cysteine string protein-α increases the rate of regulated exocytosis by modulating fusion pore dynamics in PC12 cells. PLoS One 9:e99180

    Article  Google Scholar 

  21. Yang HJ, Chen PC, Huang CT et al (2021) The phosphoprotein synapsin Ia regulates the kinetics of dense-core vesicle release. J Neurosci 41:2828–2841

    Article  CAS  Google Scholar 

  22. Han X, Wang CT, Bai J et al (2004) Transmembrane segments of syntaxin line the fusion pore of Ca2+−triggered exocytosis. Science (New York, N.Y.) 304:289–292

    Article  CAS  Google Scholar 

  23. Han X, Jackson MB (2006) Structural transitions in the synaptic SNARE complex during Ca2+−triggered exocytosis. J Cell Biol 172:281–293

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We thank Dr. Thomas F. J. Martin (University of Wisconsin-Madison) for advice in PC12 cell culture and permeabilization; Dr. Meyer B. Jackson (University of Wisconsin-Madison) for technical support in single-vesicle amperometry; Dr. Payne Y. Chang for the amperometry analysis software; the staff of Technology Commons, College of Life Science, NTU, for help with confocal microscopy; and Dr. Juu-Chin Lu and members of the Wang lab for help and discussion. This work was supported by NTU (NTU-CC-111 L891102) and the Ministry of Science and Technology (MOST-109-2311-B-002-008-MY3) to CTW. PCC is the recipient of NTU fellowship direct to advanced study for the doctoral program.

Author information

Authors and Affiliations

  1. Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan

    Pin-Chun Chen & Chih-Tien Wang

Authors
  1. Pin-Chun Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chih-Tien Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Chih-Tien Wang .

Editor information

Editors and Affiliations

  1. Department of Physical Medicine and Pharmacology, Universidad de La Laguna, La Laguna, Spain

    Ricardo Borges

Rights and permissions

Reprints and permissions

Copyright information

© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Chen, PC., Wang, CT. (2023). Rat Pheochromocytoma PC12 Cells in Culture. In: Borges, R. (eds) Chromaffin Cells. Methods in Molecular Biology, vol 2565. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2671-9_1

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-2671-9_1

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2670-2

  • Online ISBN: 978-1-0716-2671-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation